One of the first alternative transcripts characterized in the laboratory featured an in-frame link between exon 2 and 16 (band 1.8 in Table 1), excluding exons 3-15, four of the six Cu-binding sites, six of the eight transmembrane domains, and the CPC channel. The putative protein product, a 57-kDa protein, has one Cu-binding site (coded by exon 2), the phosphorylation site, the ATP-bind-ing site, and two transmembrane domains (6). Based on Western blot analysis, a protein with the calculated molecular mass of ATP7A2-16 was visible in extracts from undifferentiated BeWo cells, but was not seen when the cells were induced to differentiate (6). A movement toward differentiation restored Cu efflux function to the undifferentiated BeWo cells, reversing Cu accumulation (8). The detection of the 57-kDa protein in undifferentiated cells correlated with the expression of ATP7A2-16 mRNA. Perhaps, of interest was the observation that ATP7A2-16 mRNA is expressed in liver hepa-toma (HepG2) cells. Based on Northern analysis, others have concluded that ATP7A mRNA is not strongly expressed in liver tissue (9,10). The data suggest a tissue-specific and developmentally specific suppression of ATP7A mRNA and a concomitant augmented expression of ATP7A2-16 mRNA.

A puzzling observation was the consistent and reproducible occurrence of seven nonconforming bases in the open reading frame of ATP7A2-16 . The substitutions were cDNAs cloned from a number of human cells. This observation supports the suggestion that ATP7A mRNA may not be the parent structure of ATP7A2-16 (i.e., the truncated mRNA is not a spliced product of the larger mRNA). Currently, the origin of ATP7A2-16 is unknown, but the possibility of the mRNA arising

Fig. 1. Schematic diagram of ATP7A2-16 and NML45. Both alternative transcript products feature one Cu-binding site. NML45 is characterized by a nuclear localization sequence on the N-terminus and a -SH rich region near the C-terminus of the peptide.

nml4 5 1 mrklsirkrpnnllkecneeikmdpsmg|nsvti| Hahl i ----------------------------|fB::H

iHBBaARiBaEAgpp sg


IggBaeaSs rvInügB

copZ 32 J^jQ0KKE^v|KF\ean—VQATE®QAINE|g0Q|eVI---

NML45 61 ¡:i8BsilEEigNgTHlr*ABIWFGFAAilDGB^SN^CFlcFcSTFFSSJl Hahl 31 YD|D0PN[3gvC0ESgSSMD-TLj3z\T|[email protected]|[email protected]|LE----

Fig. 2. Amino acid sequence alignment of NML45 with copZ and Hahl. Residues showing identity in position in black. Gray indicates conservative substitutions.

from a second gene locus or genetic polymorphism cannot be dismissed. Stronger evidence for another MNK gene locus is discussed next.

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